Cross-linking of antibodies constitutes a widespread initiation signal for their respective effector functions. Cross-linking IgE-class antibodies provide the triggering signal to mast cells for their degranulation process. To obtain a quantitative insight into these cross-linking processes, the interactions between a DNP-specific monoclonal antibody of the IgE class and a series of divalent DNP haptens with spacers of different length and flexibility have been studied by fluorescence titration experiments. These were analyzed by employing the theoretical model developed by Dembo and Goldstein [Dembo, M., & Goldstein, B. (1978) J. Immunol. 121, 345-353] in a fitting procedure. Equilibrium constants that describe the aggregation and ring-closure processes caused by divalent hapten binding have been used as free parameters. The intrinsic binding constants were determined by fluorescence titrations with corresponding monovalent haptens. The main results are the following: (1) The divalent haptens with a short and flexible spacer [i.e., N alpha, N epsilon-di-(DNP)-L-lysine,meso-bis[(DNP-beta-Ala)amino]succinate, and bis[(DNP-tri-D-Ala)amino]heptane, having a maximal DNP-DNP distance of gamma = 14, 21, and 45 A, respectively] effect aggregation of the antibodies mainly into closed dimers. (2) The divalent hapten family with long and rigid oligoproline spacers di(DNP)-Ahx-Asp-(Pro)n-Lys with n = 24, 27, and 33 (i.e., gamma = 100, 110, and 130 A) causes aggregation of the antibodies predominantly into closed dimers and trimers. The corresponding equilibrium constants of the respective ring-closure processes decrease significantly with longer spacer length. (3) Evidence was found that intramolecularly monomeric ring closure of the IgE antibodies is caused by haptens containing oligoproline spacers with n = 37 or 42 (gamma = 130-150 A). The equilibrium constant of the ring-closure process increases with spacer length. This increase in stability indicates a difference in the imposed strain. Furthermore, the latter results imply that the distance between the two binding sites of the IgE molecule lies in the range dictated by the rigid oligoproline part of the respective hapten's spacer, i.e., 115-130 A. (4) Nearly all oligomeric ring-closure processes proceed relatively slowly with an approximate lower limit of a half-life of 5-10 s. This slowing down of the aggregation and ring-closure processes most probably reflects steric factors.

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BALB/c mice were repeatedly immunized with microgram doses of benzylpenicilloylated Ascaris protein(s) (BPO9Asc) in alum. At different stages of the immune response, BPO21 eicosa-L-lysine or two analogs containing one or two hydrophobic p-oxymethylbenzyl-3 beta-cholestanyl succinate (OSuco) groups were injected. When injected early in the immune response, the anti-BPO IgE antibody formation was much more strongly and permanently suppressed by the lipophilic conjugates than by the hydrophilic BPO21 eicosa-L-lysine. A similar, but less marked, suppressive effect was observed on the anti-BPO IgG1 response. By adoptive cell transfer experiments, it was found that the OSuco-containing derivatives induce and act via suppressor T lymphocytes, since this cell-mediated suppression was sensitive to cyclophosphamide or to treatment with anti-Lyt-2.2 antibody plus complement. When these compounds were injected into repeatedly immunized mice producing late ongoing antibody responses no differences in suppression between hydrophilic and hydrophobic derivatives were observed. In this case, the IgE response was suppressed by about 50%, while the IgG1 response was not affected. These results are compatible with the suggestion that early IgE responses are most sensitive to T cell-mediated suppression and that T suppressor cells are better induced by lipophilic than by hydrophilic antigens. The late ongoing IgE response, on the other hand, is less amenable to T cell-induced suppression and tolerogenic effects brought about by plurivalent BPO antigens operate directly on hapten-specific IgE-bearing B cells, regardless of their lipophilic character.

SummaryThe thiocyanate cleavage of the N"-o-nitrophenylsulfenyl group from peptides in solution or on a solid support proceeds effectively in the presence of (2-methyl-1 -indolyl)acetic acid. This scavenger was prepared from 2-methylindole and sodium bromoacetate; it can readily be removed by extraction with base after the cleavage reaction, together with (2-methyl-3-(2-nitrophenylthio)-1 -indolyl)acetic acid.The o-nitrophenylsulfenyl (Nps) group introduced into peptide synthesis by Zervas et al. [ 11 has considerable potentialities as a temporary protecting group for the a -amino terminus because its selective removal can be accomplished by nucleophilic reagents. These reagents avoid problems encountered with protecting groups requiring acids for their cleavage, i.e. alkylation of the side chains of methionine, tyrosine and also lysine and histidine by carbocations or their reaction products with the deprotecting reagent (e.g. t-butyl trifluoroacetate) [2]. Thiolytic cleavage of the Nps group with a number of reagents has been described, and recently 2-mercaptopyridine was shown to enable rapid deprotection 131 [4].In our hands the method of Wunsch & Spangenberg [ 5 ] using thiocyanate (rhodanide) in the presence of 2-methylindole proved very valuable for the cleavage of Nps-protected peptides. Thiocyanate ion attacks N-terminally bound Nps and reversibly forms o-nitrophenylsulfenyl thiocyanate (1). The equilibrium is fully displaced in the presence of excess 2-methylindole since 1 is converted into stable 3-(2-nitrophenylthio)-2-methylindole (2) and thiocyanate ion. The indole derivative is removed by washing with ether. However, with relatively lipophilic peptides, ether extraction becomes unsatisfactory. Since the two-phase-purification method of peptide synthesis [6] [7] frequently used in our laboratory depends on lipophilic peptide intermediates, it seemed worthwhile to adapt the indole reagent used in [5] and to investigate carboxy derivatives of indole which can be removed by aqueous base after reaction with an Nps residue.Using Nps-Lys(Boc)-OH as a model, the cleavage rate of a variety of reagents was assessed (Table I). It is obvious that the 3-position of the indole system should be reserved for the Nps capture and cannot be blocked as in the indoles 3-5. The

A solid‐state and solution analysis of the homo‐oligopeptides from ε‐tert.‐butyloxycarbonyl‐L‐lysine with p‐oxymethylbenzylcholestan‐3β‐yl succinate as C‐terminal group, using infrared absorption and circular dichroism, is described. The occurrence of intermolecular β‐structure is seen in the solid state and in solvents of low polarity, e.g. methylene chloride, for peptides of intermediate size (from pentamer to decamer). Conversely, the eicosapeptide exhibits a high percentage of α‐helical structure both in the solid state and in 2, 2, 2‐trifluoroethanol. The influence of the C‐terminal group on the conformational preferences of the ε‐blocked homo‐oligolysines in the solid state and in organic solvents appears negligible.